Human V?9V?2 T cells are the major subset of ?? T cells that are found in human blood, comprising up to 5% of the T cells in healthy individuals and expanding to 20-50% during infection or disease. These cells play important roles in mediating immunity against microbial pathogens, including Mycobacterium tuberculosis and Mycobacterium leprae (the causative agents of tuberculosis and leprosy, respectively), and can respond potently against certain types of tumor cells. V?9V?2 T cells respond to these threats through recognition of structurally related non-peptidic phosphorylated antigens (pAgs). In tumor cells these are intermediate metabolites that accumulate due to over-production of the mevalonate pathway and in microbes they are generated during isoprenoid biosynthesis. It is largely unknown how V?9V?2 T cells recognize these pAgs; this proposal seeks to improve our overall understanding of the mechanisms behind V?9V?2 T cell activation and improve our ability to modulate this population in clinical settings. These cells also represent a prime opportunity to study alternative recognition strategies by ?? T cells, as classical and most no-classical MHC molecules do not appear to be involved in the recognition process. Our collaborators were the first to define the role of the BTN3A molecules in pAg-mediated V?9V?2 T cell stimulation and we have since then demonstrated the intracellular domain of one on them, BTN3A1, is the molecular sensor for pAg accumulation. We seek to understand the events that occur after pAg binding in our Aim1: To determine the direct molecular consequences of pAg binding to the BTN3A1 B30.2 intracellular domain. by using structural and dynamic studies including crystallography, NMR and fluorescence based measurements (FRET). In our Aim 2. To determine the supra-molecular organization of BTN3A1 molecules in the plasma membrane upon pAg or 20.1 mAb binding and its role in V?9V?2 T cell activation, we will use FRET based approaches, microscopy and protein engineering to study the factors that mediate cell surface assembly of BTN3A molecules. Finally, we will employ cross-linking and pull-downs combined with a SILAC-based mass spectrometry approach, complemented with a high-throughput genome-wide knockdown screen to pursue our Aim 3. From BTN3A1 to the V?9V?2 TCR: discovery of the relevant molecular players linking BTN3A1 to V?9V?2 T cell activation, which will define the other molecular players that are involved in pAg recognition in inducing the signals that directly lead to V?9V?2 T cell activation.